Several pathogenic bacteria, including Listeria monocytogenes, use an F-actin-dependent motility process to spread between mammalian cells. Actin ‘comet tails’ propel Lm through the cytoplasm, resulting in bacteria-containing membrane protrusions that are internalized by neighboring cells. The mechanism by which L. monocytogenes overcomes cortical membrane tension to generate protrusions is unknown. In this work, I identify bacterial and host proteins that directly regulate the formation of protrusions. First, I show that efficient cell-cell spread in polarized epithelial cells requires the secreted Lm virulence protein, InlC. I next identify the mammalian adaptor protein Tuba as a ligand of InlC. InlC binds to a C-terminal SH3 domain in Tuba, which normally engages the human actin regulatory protein N-WASP. InlC promotes protrusion formation by inhibiting Tuba and N-WASP function, most likely by impairing binding of N-WASP to the Tuba SH3 domain. Tuba and N-WASP are known to control the structure of apical junctions in epithelial cells [1]. I demonstrate that, by inhibiting Tuba and N-WASP, InlC transforms taut apical cell-cell junctions into structures with a “slack” morphology. Experiments with Myosin II inhibitors indicate that InlC-mediated perturbation of cell junctions accounts for the role of this bacterial protein in protrusion formation. Collectively, my results suggest that InlC enhances bacterial dissemination by relieving cortical tension in apical junctions, thereby enhancing the ability of motile bacteria to deform the plasma membrane into protrusions to allow their spread into neighbouring cells.

Listeria monocytogenes has been found to withstand harsh environmental conditions including desiccation. The pathogen is also known to form biofilm when in co-culture with other bacteria found in food products. This study investigated the desiccation survival of L. monocytogenes in mixed biofilms with Pseudomonas fluorescens, Serratia liquefaciens and Shewanella putrefaciens. To this end, mono- or binary species biofilms were formed and desiccated (43% relative humidity, 21 days at 15°C) on stainless steel coupons and the double Weibull model was fitted to the resulting survivor curves. The presence of the competitor Gram-negative food spoilage bacteria with the exception of Sh. putrefaciens suppressed (p<0.05) L. monocytogenes during biofilm formation (100% relative humidity, 15°C and 48 h) and subsequently decreased (P<0.05) the desiccation survival in L. monocytogenes without affecting the resistance of individual cells. Microscopic approaches revealed different biofilm forming capabilities in the mono- and binary bacterial combinations.

The food processing industry requires alternative methods for detecting the foodborne pathogen Listeria monocytogenes that are cheaper and faster than the current methods. Conventional antibodies and their fragments have been used as biorecognition elements in sensors before, but their use is hindered by high production cost and relative instability. These issues are resolved by VHH fragments, derived from the heavy chain-only antibodies found in Camelidae. VHHs are inexpensive to produce, and are more resistant to environmental stressors. This work describes the isolation of phage-displayed VHHs that recognize recombinant Internalin B, a virulence factor characteristic of L. monocytogenes. Clone R303 was chosen for further characterization, and shown to bind full-length Internalin B. Furthermore, immobilized R303 was shown to capture L. monocytogenes cells. This panel of VHHs, particularly R303, can be utilized by colleagues within the Sentinel Bioactive Paper Network to make a viable biosensor for L. monocytogenes. / Sentinel Bioactive Paper Network

No description available.

A rapid detection method combining immunomagnetic separation (IMS), PCR and slot blot detection was developed for the detection of Salmonella and Listeria monocytogenes in milk. Bacteria were first isolated and concentrated from phosphate-buffered saline (PBS) or milk by IMS. After extraction from diluted bacteria culture with the extraction buffer, bacterial DNA was subjected to PCR. Slot blot assay was optimized and used to measure PCR products. The lowest level of detection by this method was 40 cfu/ml in PBS or milk for both pathogens. The whole detection procedure could be completed within 7 h. Moreover, this detection method is simple and easy to handle for a large number of samples. Using multiplex PCR (amplification of two different bacterial DNA in the same PCR tube) and slot blot, simultaneous detection of both bacteria was also assessed. The detection sensitivities of 103 cfu/ml for both bacteria were the same as when PCR and slot blot were used for each bacterium separately. The combination of IMS, PCR and slot blot seems to give a highly sensitive and time-efficient procedure, which could be used for routine detection of Salmonella and L. monocytogenes in milk.

Several pathogenic bacteria, including Listeria monocytogenes, use an F-actin-dependent motility process to spread between mammalian cells. Actin ‘comet tails’ propel Lm through the cytoplasm, resulting in bacteria-containing membrane protrusions that are internalized by neighboring cells. The mechanism by which L. monocytogenes overcomes cortical membrane tension to generate protrusions is unknown. In this work, I identify bacterial and host proteins that directly regulate the formation of protrusions. First, I show that efficient cell-cell spread in polarized epithelial cells requires the secreted Lm virulence protein, InlC. I next identify the mammalian adaptor protein Tuba as a ligand of InlC. InlC binds to a C-terminal SH3 domain in Tuba, which normally engages the human actin regulatory protein N-WASP. InlC promotes protrusion formation by inhibiting Tuba and N-WASP function, most likely by impairing binding of N-WASP to the Tuba SH3 domain. Tuba and N-WASP are known to control the structure of apical junctions in epithelial cells [1]. I demonstrate that, by inhibiting Tuba and N-WASP, InlC transforms taut apical cell-cell junctions into structures with a “slack” morphology. Experiments with Myosin II inhibitors indicate that InlC-mediated perturbation of cell junctions accounts for the role of this bacterial protein in protrusion formation. Collectively, my results suggest that InlC enhances bacterial dissemination by relieving cortical tension in apical junctions, thereby enhancing the ability of motile bacteria to deform the plasma membrane into protrusions to allow their spread into neighbouring cells.

Cancer vaccines can fail despite the induction of large numbers of CD8+ T cells. Two categories of memory CD8+ T cells have been defined; central memory (TCM, IL-7RαhighCD44highCD62Lhigh) and effector memory (TEM, IL-7RαhighCD44highCD62Llow). It is clear that the memory phenotype of CD8+ T cells can affect vaccine potential; however methods to augment a beneficial phenotype are not clear. I have compared three vaccine delivery systems: Listeria monocytogenes, Salmonella enterica serovar Typhimurium and the particulate liposomal adjuvant, archaeosomes, for their efficacy to protect against murine melanoma. My study revealed that the anti-tumour response is strongly influenced by the kinetics, phenotype, and lymph node homing potential of CD8+ T cells. Listeria monocytogenes-ovalbumin (LM-OVA) induced TCM cells were adept at long lasting protection against B16-OVA melanoma due to their increased homeostatic and antigen-induced proliferation, interleukin-2 production, and ability to extravasate into tumour draining lymph nodes. Conversely, although Salmonella Typhimurium-ovalbumin (ST-OVA) induced TEM, produced IFN-γ, and killed target cells, this was insufficient for long-term tumour protection. Selectin-ligand engagements of TCM cells influenced their homing potential and efficacy against murine melanoma. Fucosyltransferase deficient (FtDKO) mice, lacking functional selectin ligands, were vaccinated with LM-OVA; despite the activation of cytotoxic CD8+ T cells, there was a reduced protection against murine melanoma compared to wild-type. FtDKO CD8+ T cells exhibited reduced extravasation into FtDKO lymph nodes compared to wild-type. Additionally, fewer FtDKO CD8+ T cells compared to wild-type migrated into tumour sites. Archaeosome vaccination was used to compare the influence of CD8+ T cell quantity versus phenotype. Single or multiple therapeutic vaccinations with archaeosome-OVA yielded transient melanoma tumour protection, despite an increased frequency of circulating and tumour infiltrating CD8+ T cells. This correlated with increased expression of Program death receptor-1 (PD-1) on CD8+ T cells and induction of regulatory T cells. Prophylactic archaeosome-OVA vaccination resulted in a maximal frequency of antigen-specific CD8+ T cells of ~50-60 % with just three injections, and ~50 % of the mice were of mice were afforded long-term tumour protection (> 90 days). Overall, my study shows that the choice of vaccine adjuvant and/or vector can profoundly influence CD8+ T cell quality and cancer vaccine efficacy.

Growth and survival of L. innocua and TAC in artificially inoculated Turkish White Cheese during manufacturing and storage periods, with respect to different level of contamination of L. innocua were investigated. Cheese products were manufactured by the short-set procedure in pilot-plant-sized vats, as in AO&Ccedil / dairy factory. Pasteurized cow&rsquo / s milk was inoculated with L. innocua for obtaining the initial loads of 3.84 and 7.12 log CFU/ml. Bacterial load of inoculated milk, whey, post-ripened curd and post-salted cheese was determined during processing at 20&plusmn / 5&ordm / C. Cheeses were stored in 16% saline solution at 4 &plusmn / 2&ordm / C for up to 45 days. Samples were taken from each treatment and analysed on 5, 10, 15, 20, 30 and 45 days. Total decrease of L. innocua in Turkish White Cheese with each inoculum dose was approximately 2 logs during the storage period. L. innocua values were also compared with TAC values. The results had shown that, if pasteurization is not as sufficient as to kill this bacteria in contaminated raw milk, or if there is post-process contamination, Listeria can survive during the manufacture and storage, although they decrease in number. Storage (ripening) period for consumption of cheeses should be at least 90 and 178 days, in low and high inoculum dose, respectively. Physico-chemical properties of cheese as pH, acidity, salt, fat, moisture contents during storage period were determined. Salt concentration, pH value and storage temperature had a cumulative bactericidal effect on microorganisms. In this respect, effect of implementing HACCP method on reducing the Listerial contamination of Turkish White Cheese was determined for checking the quality problems in a cheese plant and for directing the companies as a guide.

Graduation date: 1993

Errata and corrections pasted on front end paper. / Copies of three of author's previously published articles contained in back pocket. / Bibliography: leaves 178-219. / ix, 219, [130] leaves, [31] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis aims to generate a library of chromosomally derived transcriptional promoter ::lacZ fusion mutants in an environmental isolate of L. monocytogenes (DRDC8). A lacZ transcriptional fusion mutant that displayed increased B-galactosidase activity in response to the calcium chelater EGTA is investigated in detail. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1997